Synthesis of lacto-N-neotetraose and lacto-N-tetraose using the dimethylmaleoyl group as amino protective group.

The disaccharide donor O-[2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-(1-->4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido - alpha,beta-D-glucopyranosyl] trichloroacetimidate (7) was prepared by reacting O-(2,3,4,6-tetra-O-acetyl- alpha-D-galactopyranosyl) trichloroacetimidate with tert-butyldimethylsilyl 3,6-di-O-benzyl-2-deoxy-2- dimethylmaleoylamido-glucopyranoside to give the corresponding disaccharide 5. Deprotection of the anomeric center and then reaction with trichloroacetonitrile afforded 7. Reaction of 7 with 3'-O-unprotected benzyl (2,4,6-tri-O-benzyl-beta-D-galactopyranosyl)- (1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside (8) as acceptor afforded the desired tetrasaccharide benzyl (2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->4)-(3,6-di-O- benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl)-(1-->3)- (2,4,6- tri-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-beta-D- glucopyranoside. Replacement of the N-dimethylmaleoyl group by the acetyl group, O-debenzylation and finally O-deacetylation gave lacto-N-neotetraose. Similarly, reaction of O-[(2,3,4,6-tetra-O-acetyl-beta- D-galactopyranosyl)-(1-->3)-4,6-O-benzylidene-2-deoxy-2-dimethylmalei mido- alpha,beta-D-glycopyranosyl] trichloroacetimidate as donor with 8 as acceptor afforded the desired tetrasaccharide benzyl (2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-(1-->3)-(4,6-benzylidene-2-deoxy-2-dimethylmaleimid o- beta-D-glucopyranosyl)-(1-->3)-(2,4,6-tri-O-benzyl-beta-D-galactopyranos yl)- (1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside. Removal of the benzylidene group, replacement of the N-dimethylmaleoyl group by the acetyl group and then O-acetylation afforded tetrasaccharide intermediate 15, which carries only O-benzyl and O-acetyl protective groups. O-Debenzylation and O-deacetylation gave lacto-N-tetraose (1). Additionally, known tertbutyldimethylsilyl (2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->3)-4,6-O-benzylide ne- 2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside was transformed into O-[2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)- (1-->3)-4,6-di-O-acetyl-2-deoxy-2-dimethylmaleimido-alpha,beta-D- glucopyranosyl] trichloroacetimidate as glycosyl donor, to afford with 8 as acceptor the corresponding tetrasaccharide 22, which is transformed into 15, thus giving an alternative approach to 1.     

Facile preparation of deoxyiodocellulose and its conversion into 5,6-cellulosene

Treatment of 6-chloro-6-deoxycellulose with sodium iodide in 2,5-hexanedione gave 6-deoxy-6-iodocellulose; >80% of the chlorine atoms were replaced. 6-Deoxy-6-iodo-d-glucose was identified by g.l.c.-m.s. in hydrolyzates of deoxyiodocellulose. Acetylated 6-deoxy-6-iodocellulose was converted almost quantitatively into 5,6-cellulosene acetate, which was characterized by hydrolyzing the product and converting the resultant dicarbonyl sugar into an isopropylidene acetal 10. The changes of molecular-weight distribution during iodination and dehydroiodination were investigated by gel-permeation chromatography.     

An enzymatic microassay for lactose

An enzymatic microassay for lactose using a lactase enzyme derived from Saccharomyces fragilis is described. The assay uses 50-μl samples, provides 100% hydrolysis of lactose, and is sensitive within the range of 12.5–500 nmol per sample. The assay has been validated against an assay for ¹⁴C lactose which involves thin-layer chromatographic isolation of lactose. The assay is sufficiently sensitive for use in physiologic studies.     

Study of glycosylation with N-trichloroacetyl-D-glucosamine derivatives in the syntheses of the spacer-armed pentasaccharides sialyl lacto-N-neotetraose and sialyl lacto-N-tetraose, their fragments, and analogues.

The syntheses of 2-aminoethyl glycosides of the pentasaccharides Neu5Ac-alpha(2-->3)-Gal-beta(1-->4)-GlcNAc-beta(1-->3)-Gal-beta(1-->4)-Glc and Neu5Ac-alpha(2-->3)-Gal-beta(1-->3)-GlcNAc-beta(1-->3)-Gal-beta(1-->4)-Glc, their asialo di-, tri-, and tetrasaccharide fragments, and analogues included a systematic study of glycosylation with variously protected mono- and disaccharide donors derived from N-trichloroacetyl-D-glucosamine of galactose, lactose, and lactosamine glycosyl acceptors bearing benzoyl protection around the OH group to be glycosylated. Despite the low reactivity of these acceptors, stereospecificity and good to excellent yields were obtained with NIS-TfOH-activated thioglycoside donors of such type, or with AgOTf-activated glycosyl bromides, while other promotors, as well as a trichloroacetimidate donor, were less effective, and a beta-acetate donor was inactive. In NIS-TfOH-promoted glycosylation with the thioglycosides, the use of TfOH in catalytic amount led to rapid formation of the corresponding oxazoline, but the quantity of TfOH necessary for further efficient coupling with an acceptor depended on the reactivity of the donor, varying from 0.07 equiv for a 3,6-di-O-benzylated monosaccharide derivative to 2.1 equiv for a peracetylated disaccharide one. In the glycosylation products, the N-trichloroacetyl group was easily converted into N-acetyl by alkaline hydrolysis followed by N-acetylation.     

In vitro enzymatic conversion of pantothenylcysteine-4'-phosphate into cysteamine.

In vivo production of taurine can not proceed from cysteine via the action of cysteinsulphinic acid-decarboxylase in those mammalian organs where this enzymatic activity is absent, such as heart. The possibility of existence of another metabolic way is strengthened by the experimental finding that, in vitro, cysteamine is obtained from pantothenylcysteine-4'-phosphate through the combined action of two mammalian enzymes, i.e. pantothenylcysteine-4'-phosphate decarboxylase and pantetheinase, used in partially purified forms. This new route should be related to the known biosynthetic pathway of CoA.     

Product inhibition of the enzymatic hydrolysis of lactose

Kinetic experiments have been conducted with acetone-dried cells of Kluyveromyces fragilis to study product inhibition of the enzymatic hydrolysis of lactose. Both hydrolytic products, d-glucose and d-galactose, showed efficient inhibition effect on enzyme activity. The fact that d-glucose and d-galactose are mutually exclusive for the inhibition was verified by Dixon plots. The kinetic constants were also estimated using the experimental data. The rate equation was derived based on a multiple inhibition model of competitive inhibition of d-galactose and non-competitive inhibition of d-glucose. The good agreement between experiment and prediction indicated the validity of the established model.     

Development of an ultrahigh-temperature process for the enzymatic hydrolysis of lactose. IV. Immobilization of two thermostable beta-glycosidases and optimization of a packed-bed reactor for lactose conversion.

Recombinant hyperthermostable beta-glycosidases from the archaea Sulfolobus solfataricus (Ss beta Gly) and Pyrococcus furiosus (CelB) were covalently attached onto the insoluble carriers chitosan, controlled pore glass (CPG), and Eupergit C. For each enzyme/carrier pair, the protein-binding capacity, the immobilization yield, the pH profiles for activity and stability, the activity/temperature profile, and the kinetic constants for lactose hydrolysis at 70 degrees C were determined. Eupergit C was best among the carriers in regard to retention of native-like activity and stability of Ss beta Gly and CelB over the pH range 3.0-7.5. Its protein binding capacity of approximately 0.003 (on a mass basis) was one-third times that of CPG, while immobilization yields were typically 80% in each case. Activation energies for lactose conversion by the immobilized enzymes at pH 5.5 were in the range 50-60 kJ/mol. This is compared to values of approximately 75 kJ/mol for the free enzymes. Immobilization expands the useful pH range for CelB and Ss beta Gly by approximately 1.5 pH units toward pH 3.5 and pH 4.5, respectively. A packed-bed enzyme reactor was developed for the continuous conversion of lactose in different media, including whey and milk, and operated over extended reaction times of up to 14 days. The productivities of the Eupergit C-immobilized enzyme reactor were determined at dilution rates between 1 and 12 h(-1), and using 45 and 170 g/L initial lactose. Results of kinetic modeling for the same reactor, assuming plug flow and steady state, suggest the presence of mass-transfer limitation of the reaction rate under the conditions used. Formation of galacto-oligosaccharides in the continuous packed-bed reactor and in the batch reactor using free enzyme was closely similar in regard to yield and individual saccharide components produced.     

Optimal design for CSTR's in series performing enzymatic lactose hydrolysis

Analytical expressions are derived for the optimal design (based on minimum overall reactors volume) of a series of N CSTR's performing enzymatic lactose hydrolysis. It is assumed that lactose hydrolysis obeys Michaelis-Menten kinetics with competitive product (galactose) inhibition and no enzyme deactivation occurs. The optimum design of a cascade of ideally mixed reactors are compared with equal size reactors and with plug flow reactor required for a given overall degree of lactose conversion. The effect of operating parameters such as temperature, lactose initial (feed) concentration and conversion, enzyme and product initial concentration on the optimal overall holding time are also investigated. Optimization results for a series of N CSTR's up to five are obtained and compared with plug flow reactor.     

New type of glucose sensor based on enzymatic conversion of gel volume into liquid column length

A way to convert the volume change of a biochemo-mechanical gel into the change in liquid column length was developed. Our trial sensor device consisted of a small compartment for incorporating the gel, a flow channel with a filled dye solution, and a poly(dimethylsiloxane) (PDMS) diaphragm by which the gel and the dye solution were separated. A lightly cross-linked N-isopropylacrylamide (NIPAAm)/acrylic acid (AA) copolymer gel with immobilized glucose oxidase was used as a sensing element. It was found that a change in the gel volume caused by the immobilized enzyme reaction was accurately converted into a change of the column length (Deltal) with the help of the PDMS diaphragm. By use of a cylindrical gel (diameter approximately 2 and thickness approximately 1 mm), the time curve of Deltal varied depending upon glucose concentration over a range of 0.2-50 mM; in particular, it is of importance that semilogarithmic plots of Deltal (in mm) against glucose concentration (in mM) can be used as a calibration curve. For glucose solutions of mM order, 1 min was enough to determine the concentrations, whereas 10 min was required for concentrations of microM order. When the measurement time was limited within 10 min, the lower detection limit was 200 microM. The response was affected by buffering capacity of the samples, but this was controllable through reduction of the sample volume. These results indicate that the present way can be used for the determination of glucose concentration.     

The enzymatic conversion of arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid. Resolution of rat lung enzyme into two active fractions

The conversion of arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid by rat lung high-speed supernatant has been resolved into two separate stages through ammonium sulfate precipitation. The first stage is catalysed by 0-30% ammonium sulfate fraction and converts arachidonic acid and 12-hydroperoxyeicosatetraenoic acid into an intermediate, X. X is subsequently utilized in the second stage by the fraction sedimented at 30-50% saturation in ammonium sulfate to form two isomeric 8,11,12-trihydroxyeicosatrienoic acids.     

Further studies on the enzymatic conversion of prostaglandin endoperoxide into prostacyclin by porcine aorta microsomes.

A simple, rapid radiochemical assay for prostacyclin synthesis has been used to characterize the enzyme in arterial walls which converts prostaglandin endoperoxides to prostacyclin. The enzyme displays a broad pH optimum, and catalyses a rapid conversion of saturating concentrations of the endoperoxide at 37 degrees C. Hydroperoxides of several unsaturated fatty acids are potent inhibitors of the enzyme, and act in a time dependent manner. The isomerase which converts prostaglandin endoperoxides to prostaglandin E2 or D2 was not detected in the arterial wall.     

Mechanisms of lactose utilization by lactic acid streptococci: enzymatic and genetic analyses

The apparent instability of beta-galactosidase in toluene-treated cells or cell-free extracts of lactic streptococci is explained by the fact that these organisms do not contain the expected enzyme. Instead, various strains of Streptococcus lactis, S. cremoris, and S. diacetilactis were shown to hydrolyze o-nitrophenyl-beta-d-galactoside-6-phosphate (ONPG-6-P), indicating the presence of a different enzyme. In addition, lactose metabolism in S. lactis C(2)F was found to involve enzyme I (EI), enzyme II (EII), factor III (FIII), and a heat-stable protein (HPr) of a phosphoenolpyruvate (PEP)-dependent phosphotransferase system analogous to that of Staphylococcus aureus. Mutants of S. lactis C(2)F, defective in lactose metabolism, possessed the phenotype lac(-) gal(-). These strains were unable to accumulate (14)C-thiomethyl-beta-d-galactoside, to hydrolyze ONPG, or to utilize lactose when grown in lactose or galactose broth. In addition, these mutants contained EI and HPr, but lacked EII, FIII, and the ability to hydrolyze ONPG-6-P. This suggested that the defect was in the phosphorylation step. Lactose-negative mutants of S. lactis 7962, a strain containing beta-galactosidase, could be separated into several classes, which indicated that this organism is not dependent upon the PEP-phosphotransferase system for lactose metabolism.